National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland.
National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland.
Curr Protoc. 2021 Oct;1(10):e276. doi: 10.1002/cpz1.276.
Cyclin-dependent kinases (Cdks) are generally known to be involved in controlling the cell cycle, but Cdk5 is a unique member of this protein family for being most active in post-mitotic neurons. Cdk5 is developmentally important in regulating neuronal migration, neurite outgrowth, and axon guidance. Cdk5 is enriched in synaptic membranes and is known to modulate synaptic activity. Postnatally, Cdk5 can also affect neuronal processes such as dopaminergic signaling and pain sensitivity. Dysregulated Cdk5, in contrast, has been linked to neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). Despite primarily being implicated in neuronal development and activity, Cdk5 has lately been linked to non-neuronal functions including cancer cell growth, immune responses, and diabetes. Since Cdk5 activity is tightly regulated, a method for measuring its kinase activity is needed to fully understand the precise role of Cdk5 in developmental and disease processes. This article includes methods for detecting Cdk5 kinase activity in cultured cells or tissues, identifying new substrates, and screening for new kinase inhibitors. Furthermore, since Cdk5 shares homology and substrate specificity with Cdk1 and Cdk2, the Cdk5 kinase assay can be used, with modification, to measure the activity of other Cdks as well. © 2021 Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: Measuring Cdk5 activity from protein lysates Support Protocol 1: Immunoprecipitation of Cdk5 using Dynabeads Alternate Protocol: Non-radioactive protocols to measure Cdk5 kinase activity Support Protocol 2: Western blot analysis for the detection of Cdk5, p35, and p39 Support Protocol 3: Immunodetection analysis for Cdk5, p35, and p39 Support Protocol 4: Genetically engineered mice (+ and - controls) Basic Protocol 2: Identifying new Cdk5 substrates and kinase inhibitors.
周期蛋白依赖性激酶（Cdks）通常被认为参与控制细胞周期，但 Cdk5 是该蛋白家族中一个独特的成员，因为它在有丝分裂后的神经元中最为活跃。Cdk5 在调节神经元迁移、突起生长和轴突导向方面具有重要的发育意义。Cdk5 富含突触膜，并已知可调节突触活性。出生后，Cdk5 还可以影响神经元过程，如多巴胺能信号和疼痛敏感性。相反，失调的 Cdk5 与神经退行性疾病有关，如阿尔茨海默病（AD）、帕金森病（PD）和肌萎缩侧索硬化症（ALS）。尽管 Cdk5 主要与神经元的发育和活动有关，但最近它与非神经元功能有关，包括癌细胞生长、免疫反应和糖尿病。由于 Cdk5 的活性受到严格调控，因此需要一种测量其激酶活性的方法来充分了解 Cdk5 在发育和疾病过程中的精确作用。本文包括在培养细胞或组织中检测 Cdk5 激酶活性、鉴定新底物和筛选新激酶抑制剂的方法。此外，由于 Cdk5 与 Cdk1 和 Cdk2 具有同源性和底物特异性，因此可以使用 Cdk5 激酶测定法（稍加修改）来测量其他 Cdk 的活性。© 2021 威立出版社。本文的作者是美国政府的雇员，他们的工作在美国属于公有领域。基本方案 1：从蛋白裂解物中测量 Cdk5 活性支持方案 1：使用 Dynabeads 免疫沉淀 Cdk5 备选方案 1：非放射性测定法测量 Cdk5 激酶活性支持方案 2：检测 Cdk5、p35 和 p39 的 Western blot 分析支持方案 3：Cdk5、p35 和 p39 的免疫检测分析支持方案 4：基因工程小鼠（+和-对照）基本方案 2：鉴定新的 Cdk5 底物和激酶抑制剂。
